The use of high-dose methotrexate therapy with citrovorum factor, 5-formyl-5,6,7,8-tetrahydrofolic acid, (5-CHO-THF rescue is under active investigation for the treatment of a number of solid tumors and hematologic malignancies. The development of successful protocols will result in the need for larger amounts of citrovorum factor (5-CHO-THF).
By the present invention, there are provided improved methods for the preparation and purification of citrovorum factor. In one aspect of the invention, formylation of folic acid (FA) gave 10-CHO-FA, which was hydrogenated in trifluoroacetic acid to give high yields of (5, 10-CH-THF).sup.+ , the dehydration product of the initially formed 10-CHO-THF. In another aspect of the invention, the reduction of folic acid with borohydride followed by treatment of the resulting THF with formic acid gave good yields of (5, 10-CH-THF).sup.+, isolated as the chloride. The effect of base concentration, temperature, and time of reaction on the conversion of (5, 10-CH-THF).sup.+ Cl.sup.- to 5-CHO-THF was determined. These methods led to the preparation of the calcium salt dihydrate of 5-CHO-THF in high yields, which was about 78% pure. The identification of the impurities in these 5-CHO-THF samples was determined by high-pressure liquid chromatography, and the removal of the impurities was effected by Florisil chromatography. The discovery of a nonchromatographic method for the removal of most of the impurities from crude samples of 5-CHO-THF is also described.
The decrease in toxicity and increase in therapeutic benefit resulting from the adjuvant treatment of osteogenic sarcoma with a high dose of methotrexate followed by rescue with citrovorum factor, 5-formyl-5,6,7,8-tetrahydrofolic acid, (5-CHO-THF) has been established by Jaffe et al., New Engl. J. Med., 291, 994 (1974). In this modality, 5-CHO-THF apparently protects normal sensitive tissue without canceling the inhibitory activity of methotrexate against neoplastic tissue. In addition, this form of therapy has been reported as being potentially effective against other malignancies, including refractory acute leukemia, bronchogenic carcinoma and head and neck cancer. Although the ultimate value in terms of cures of this form of therapy has not been fully documented, the development of the high-dose methotrexate regimen requires large amounts of both methotrexate and 5-CHO-THF. Recently, there has been reported by J.R. Piper and J.A. Montgomery, J. Heterocycl. Chem. 11, 279 (1974), an improved method for the large-scale synthesis of methotrexate of high purity, and we now report improved procedures for the large-scale preparation and purification of 5-CHO-THF.
The synthesis and identification of 5-CHO-THF was carried out about 25 years ago, as described, for example, by Pohland et al., J. Am. Chem. Soc., 73, 3247 (1951). In general, the adopted procedure involved the formylation of folic acid with formic acid, catalytic hydrogenation of the pyrazine ring of the resulting formic acid solution of 10-CHO-FA, and treatment of the product of the reduction with base at elevated temperatures to give crude 5-CHO-THF. In one procedure, bioassay of the crude product indicated that about a 22% yield of 5-CHO-THF was obtained. Purification was effected by column chromatography to give a low yield of 5-CHO-THF isolated as the barium salt pentahydrate. Although no yields were reported, a similar procedure was used for the preparartion of the calcium salt of 5-CHO-THF.
In the prior art synthesis described above, it was recognized that 5-CHO-THF was dehydrated under acidic conditions to give (5, 10-CH-THF).sup.+ and that the same product was formed from 10-CHO-THF resulting from the hydrogenation of 10-CHO-FA in formic acid. Treatment of (5,10-CH-THF).sup.+ with base at room temperature opened the imidazolinium ring to give mainly 10-CHO-THF (kinetic control), which underwent oxidation readily in the presence of oxygen and light to give decomposition products, as described by May et al., J. Am. Chem. Soc., 73, 3067 (1951). In contrast, treatment of (5,10-CH-THF).sup.+ with base at higher temperatures resulted in the formation of 5-CHO-THF (thermodynamic control) and decomposition products.